Immunoconjugates with an intracellularly-cleavable linkage

ABSTRACT

The invention relates to therapeutic conjugates with improved ability to target various cancer cells containing a targeting moiety and a therapeutic moiety. The targeting and therapeutic moieties are linked via an acid cleavable linkage that increases therapeutic efficacy of the immunoconjugate.

This application is a divisional of U.S. patent application Ser. No.13/845,694, filed Mar. 18, 2013, which was a divisional of U.S. patentapplication Ser. No. 13/586,281 (now U.S. Pat. No. 8,425,912), filedAug. 15, 2012, which was a divisional of U.S. patent application Ser.No. 12/512,526 (now U.S. Pat. No. 8,268,319), filed Jul. 30, 2009, whichwas a divisional of U.S. patent application Ser. No. 10/734,589, (nowU.S. Pat. No. 7,585,491), filed Dec. 15, 2003, which claimed priority toU.S. Provisional Application No. 60/433,017 filed Dec. 13, 2002, thecontents of each of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to therapeutic conjugates with improvedability to target various cancer cells, infectious disease organisms andfor treating autoimmune diseases, which conjugates contain a targetingmoiety and a therapeutic moiety. The targeting and therapeutic moietiesare linked via an intracellularly cleavable linkage that increasestherapeutic efficacy.

BACKGROUND OF THE INVENTION

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (mAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated mAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer, and virtually noapplication in other diseases. The toxic agent is most commonly achemotherapy drug, although particle-emitting radionuclides, orbacterial or plant toxins have also been conjugated to mAbs.

The advantages of using mAb-chemotherapy drug conjugates are that (a)the chemotherapy drug itself is structurally well defined; (b) thechemotherapy drug is linked to the mAb protein using very well definedconjugation chemistries, often at specific sites remote from the mAbsantigen binding regions; (c) mAb-chemotherapy drug conjugates can bemade more reproducibly than chemical conjugates involving mAbs andbacterial or plant toxins, and as such are more amenable to commercialdevelopment and regulatory approval; and (d) the mAb-chemotherapy drugconjugates are orders of magnitude less toxic than radionuclide mAbconjugates.

Relevant early work on mAb-chemotherapy drug conjugates found during invitro and in vivo preclinical testing that the chemical linkages usedoften resulted in the loss of a drug's potency. These results indicatedthat a drug ideally needed to be released in its original form, once ithad been internalized into a target cell, for the mAb-chemotherapy drugconjugate to be a useful therapeutic. Accordingly, work during the1980's and early 1990's focused largely on the nature of the chemicallinker between the chemotherapeutic drug and the mAb. Notably,mAb-chemotherapy drug conjugates prepared using mild acid-cleavablelinkers were developed, based on the observation that the pH insidetumors was often lower than normal physiological pH (U.S. Pat. Nos.4,542,225; 4,569,789; 4,618,492; and 4,952,394). This approachculminated in a landmark paper by Trail et al. (Science 261:212-215(1993)) which showed that mAb-doxorubicin (DOX) conjugates, preparedwith appropriate linkers, could be used to cure mice bearing a varietyof human tumor xenografts, in preclinical studies. This promising resultwas achieved with an antibody that bound to a very large number ofreceptors on the tumor cells being targeted, and the mAb-chemotherapydrug conjugate was substituted with six to eight DOX residues per unitof mAb. Further, the mAb-chemotherapy drug conjugate was given inmassive doses on a repeated dosage schedule.

During the development of the aforementioned mAb-chemotherapy drugconjugates, the linker between the chemotherapeutic drug and the mAb wasthought to be critical for retention of good anti-tumor activity both invitro and in vivo. In some cases, the mAb-chemotherapy drug conjugateswere made with acid-labile (e.g., hydrazone) and reductively labile(e.g., disulfide) bonds between the chemotherapy drugs and the mAb.While the hydrazone bond is apparently stable to in vivo serumconditions, normal disulfide bonds were found to be not stable enoughfor practical use. Accordingly, mAb-chemotherapy drug conjugates weredeveloped that replaced a standard disulfide linkage with a hinderedgeminal dimethyl disulfide linkage or a methyl disulfide linkage.

Other work in the field has focused on the use of a hydrazone as thecleavable moiety and a thioether group instead of a disulfide linkage.Willner et al. have found superior results by incorporating a hydrazoneas a cleavable unit, and attaching DOX to a mAb via a thioether group,instead of a disulfide (U.S. Pat. No. 5,708,146). When linked in such amanner, and using a branched linker capable of doubling the number ofDOX units per mAb substitution site, an approximate order of magnitudeincrease in the efficacy of the new DOX-mAb conjugates was obtained(King et al., Bioconjugate Chem. 10:279-288 (1999)).

Another cleavable moiety that has been explored is an ester linkageincorporated into the linker between the antibody and the chemotherapydrug. Gillimard and Saragovi have found that when an ester of paclitaxelwas conjugated to anti-rat p75 mAb, MC192, or anti-human TrkA mAb, 5C3,the conjugate was found to exhibit target specific-toxicity. Gillimardand Saragovi, Cancer Res. 61:694-699 (2001).

Realizing the importance of the linkers in the construction ofmAb-chemotherapy drug conjugates, the inventors have developed novellinkers which may be used generally with a variety of chemotherapeuticdrugs and other toxic agents.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to an immunoconjugatecomprising:

(a) a targeting moiety;

(b) a chemotherapeutic moiety; and

(c) a linker binding to the targeting moiety via a thiol group, and tothe chemotherapeutic moiety via an intracellularly-cleavable moietyother than a hydrazone. In another embodiment, the invention relates toan immunoconjugate comprising:

(a) targeting moiety;

(b) a chemotherapeutic moiety; and

(c) a linker binding to the targeting moiety via a thiol group, and tothe chemotherapeutic moiety via an intracellularly-cleavable moietyother than a hydrazone;

wherein said linker comprises an amino acid moiety and a thiol-reactivemoiety and is linked to the chemotherapeutic moiety via the amino acidmoiety and to the targeting moiety via the thiol-reactive moiety.

Another embodiment of the invention is a method of treating cancer, amalignancy, an autoimmune disease, an infection, or an infectious lesionwith the immunoconjugates described herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless otherwise specified, “a” or “an” means “one or more.”

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thepresent invention.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv) and the like. Regardlessof structure, an antibody fragment binds with the same antigen that isrecognized by the intact antibody.

The term “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the variable regions, such as the “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region. The Fv fragments may beconstructed in different ways as to yield multivalent and/ormultispecific binding forms. In the former case of multivalent, theyreact with more than one binding site against the specific epitope,whereas with multispecific forms, more than one epitope (either of theantigen or even against the specific antigen and a different antigen) isbound.

As used herein, the term antibody component includes both an entireantibody, a fusion protein, and fragments thereof.

A naked antibody is generally an entire antibody which is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector or immunological functions, such ascomplement fixation and ADCC (antibody dependent cell cytotoxicity),which set mechanisms into action that may result in cell lysis. However,the Fc portion may not be required for therapeutic function of theantibody, but rather other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,such as inhibition of heterotypic or homotypic adhesion, andinterference in signaling pathways, may come into play and interferewith the disease progression. Naked antibodies include both polyclonaland monoclonal antibodies, and fragments thereof, that include murineantibodies, as well as certain recombinant antibodies, such as chimeric,humanized or human antibodies and fragments thereof. As defined in thepresent invention, “naked” is synonymous with “unconjugated,” and meansnot linked or conjugated to the therapeutic agent with which itadministered.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, while the constant domains ofthe antibody molecule are derived from those of a human antibody. Forveterinary applications, the constant domains of the chimeric antibodymay be derived from that of other species, such as a cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, is transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule is derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated intheir entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody component,i.e., an antibody or antibody fragment, or a subfragment thereof, and isuseful in the treatment of a disease. Examples of therapeutic agentsinclude antibodies, antibody fragments, immunoconjugates, drugs,cytotoxic agents, toxins, nucleases, hormones, immunomodulators,chelators, boron compounds, photoactive agents or dyes, radioisotopes orradionuclides, immunoconjugates or combinations thereof.

An immunoconjugate is an antibody component conjugated to a therapeuticagent. Suitable therapeutic agents are described above.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which two or more ofthe same or different natural antibody, single-chain antibody orantibody fragment segments with the same or different specificities arelinked. A fusion protein comprises at least one specific binding site.

Valency of the fusion protein indicates the total number of binding armsor sites the fusion protein has to antigen(s) or epitope(s); i.e.,monovalent, bivalent, trivalent or mutlivalent. The multivalency of theantibody fusion protein means that it can take advantage of multipleinteractions in binding to an antigen, thus increasing the avidity ofbinding to the antigen, or to different antigens. Specificity indicateshow many different types of antigen or epitope an antibody fusionprotein is able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one type of antigen or epitope. A monospecific,multivalent fusion protein has more than one binding site for the sameantigen or epitope. For example, a monospecific diabody is a fusionprotein with two binding sites reactive with the same antigen. Thefusion protein may comprise a multivalent or multispecific combinationof different antibody components or multiple copies of the same antibodycomponent. The fusion protein may additionally comprise a therapeuticagent.

An immunomodulator is a therapeutic agent as defined in the presentinvention that when present, alters, suppresses or stimulates the body'simmune system. Typically, the immunomodulator useful in the presentinvention stimulates immune cells to proliferate or become activated inan immune response cascade, such as macrophages, B-cells, and/orT-cells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDsthat are released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which act as intercellular mediatorsbetween cells. As the skilled artisan will understand, examples ofcytokines include lymphokines, monokines, interleukins, and severalrelated signalling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation.

In a preferred embodiment, the intracellularly-cleavable moiety isoptionally cleavable by intracellular esterases. In a preferredembodiment, the intracellularly-cleavable moiety is an ester moiety. Ina preferred embodiment, the ester moiety is the ester formed from theα-carboxylic acid of an amino acid. In a preferred embodiment, theintracellularly-cleavable moiety comprises a peptide bond cleavable byintracellular enzymes. In a preferred embodiment, the linker comprises athiol-reactive group which links to thiol groups of said targetingmoiety. The thiol-reactive group is optionally a maleimide orvinylsulfone which links to thiol groups of said targeting moiety. In apreferred embodiment, the linker comprises a thiol group which reactswith a maleimide residue at a lysine side chain of said targetingmoiety. The immunoconjugate preferably further comprises anaminopolycarboxylate residue between the chemotherapetic moiety and thetargeting moiety. The aminopolycarboxylate is preferably selected fromthe group consisting of DTPA (diethylenetriaminepentaacetic acid), EDTA(ethylenediaminetetraacetic acid), TTHA (triethylenetetraminehexaaceticacid), benzyl-DTPA, DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid),benzyl-DOTA, NOTA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid),benzyl-NOTA and TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid).

The chemotherapeutic moiety is preferably selected from the groupconsisting of doxorubicin (DOX), epirubicin, morpholinodoxorubicin(morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX),2-pyrrolino-doxorubicin (2-PDOX), camptothecin (CPT), irinotecan(CPT-11), SN-38 (the active drug which is metabolically produced, invivo, from the prodrug CPT-11), topotecan, taxanes, geldanamycin,ansamycins, and epothilones. Targeting moiety is a monoclonal antibody(mAb).

In one embodiment, the targeting moiety is a monoclonal antibody (mAb).In a further embodiment, the targeting moiety may be a multivalentand/or multispecific mAb. The targeting moiety may be a murine,chimeric, humanized, or human monoclonal antibody, and said antibody isin intact, fragment (Fab, Fab′, F(ab)₂, F(ab′)₂), or sub-fragment(single-chain constructs) form.

In a preferred embodiment, the targeting moiety is a monoclonal antibodythat is reactive with an antigen or epitope of an antigen expressed on acancer or malignant cell. The cancer cell is preferably a cell from ahematopoietic tumor, carcinoma, sarcoma, melanoma or a glial tumor.

The targeting moiety preferably links to at least one chemotherapeuticmoiety, more preferably it links to about 7 to 12 said chemotherapeuticmoieties. A preferred linker has the formula:

The linker optionally comprises a functional group at the N-terminus, awater-solubilizing moiety at the C-terminus, and one or more internalbasic amino acids with side chains available for attachment to saidchemotherapeutic moiety. The water-solubilizing moiety is optionallyDTPA, EDTA, TTHA, benzyl-DTPA, DOTA, benzyl-DOTA, NOTA, benzyl-NOTA, orN,N′-dialkyl substituted piperazine. The functional group is optionallya thiol-reactive or an amine-reactive group.

The preferred chemotherapeutic moiety is selected from the groupconsisting of doxorubicin (DOX), epirubicin, morpholinodoxorubicin(morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX),2-pyrrolino-doxorubicin (2-PDOX), CPT, CPT-11, SN-38, topotecan,taxanes, geldanamycin, ansamycins, and epothilones.

The immunoconjugates may be used to treat a variety of diseases andconditions, including cancer, malignancies and infectious lesions. Apreferred malignancy to be treated according to the present invention isa malignant solid tumor or hematopoietic neoplasm. A preferred target ofthe immunoconjugate when the method is directed to treatment of aninfectious lesion is antigen or epitope or iron-siderophore chelatereceptor on a pathogen. The pathogen is preferably selected from thegroup consisting of a bacterium, fungus, virus, rickettsia, mycoplasmaand protozoa. In particular, the pathogen may be selected from the groupconsisting of Streptococcus agalactiae, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, mycobacterium tuberculosis,rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I,herpes simplex virus II, human serum parvo-like virus, respiratorysyncytial virus, varicella-zoster virus, hepatitis B virus, measlesvirus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus,murine leukemia virus, mumps virus, vesicular stomatitis virus, sindbisvirus, lymphocytic choriomeningitis virus, wart virus, blue tonguevirus, Sendai virus, feline leukemia virus, reo virus, polio virus,simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus,Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosomarangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosomabrucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis,Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinellaspiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taeniasaginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasmaarthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasmalaidlawii, M. salivarium and M. pneumoniae.

The immunoconjugates may also be used to treat autoimmune disease,including class III autoimmune diseases. Preferred class III autoimmunediseases for treatment according to the invention are selected from thegroup consisting of immune-mediated thrombocytopenias, dermatomyositis,Sjögren's syndrome, multiple sclerosis, Sydenham's chorea, myastheniagravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever,rheumatoid arthritis, polyglandular syndromes, bullous pemphigoid,diabetes mellitus, Henoch-Schonlein purpura, post-streptococcalnephritis, erythema nodosum, Takayasu's arteritis, Addison's disease,rheumatoid arthritis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis ubiterans, primarybiliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma,chronic active hepatitis, polymyositis/dermatomyositis, polychondritis,pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis.

The immunoconjugates are preferably administered parenterally.

In one aspect of the preferred embodiments of the present invention, theinvention relates to an immunoconjugate comprising a targeting moiety, achemotherapeutic moiety and a linker binding to the targeting moiety viaa thiol group, and to the chemotherapeutic moiety via anintracellularly-cleavable moiety other than a hydrazone.

In a preferred embodiment, the intracellularly-cleavable moiety otherthan a hydrazone are moieties that may be cleaved once internalized intothe cell to which the mAb recognizes and binds to a receptor, andparticularly cleaved by esterases and amidases.

In a preferred embodiment, the chemotherapeutic moiety is separatelyactivated at neutral pH such that it contains a thiol-reactive group. Itis within the skill of the ordinary skilled artisan to developchemistries that would allow for the incorporation of thiol-reactivegroups into any chemotherapeutic moiety. In a preferred embodiment,however, the chemotherapeutic moiety may be activated such that thegroup bearing the thiol-reactive group is spaced from thechemotherapeutic moiety by an amino acid radical. In a preferredembodiment, the α-amino group of the amino acid may reacted with areagent bearing a thiol-reactive group, such as maleimide,chloroacetamide, bromoacetamide, iodoacetamide, and vinylsulfone.Preferably, said reagent bearing a thiol-reactive group issuccinimidyl[4-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). In thecase of SMCC, the thiol-reactive group is a maleimide group. The productresulting from the reaction of SMCC and the chemotherapeutic moietycomprising an amino acid radical is the general compound illustratedbelow:

wherein R is the side chain of any amino acid.

While not wishing to be bound by this theory, the maleimido cyclohexylcarboxylate group is thought to be particularly useful in the context ofthe preferred embodiments of the present invention for two reasons.First, the cyclohexyl group in the linker is thought to stabilize theamide functionality formed from the reaction of SMCC and the amino acidα-amino group. Second, the ester formed between the drug and the aminoacid carboxylate is cleaved once the immunoconjugate is internalizedinto the cell. An example of intracellularly cleaved ester bond ispaclitaxel-MC192 immunoconjugate (Gillimard and Saragovi, supra).

In a preferred embodiment of the present invention, the chemotherapeuticmoiety comprising an amino acid radical, as shown above, contains thelinker; that is, the linker binding to the targeting moiety via a thiolgroup. The linker is the portion of the molecule shown below:

However, the scope of the preferred embodiments of the present inventionis not so limited. The linker may be a moiety that simply comprises anamino acid radical and a thiol-reactive group. The amino acid radicaland the thiol-reactive group may be spaced by any moieties known to theskilled artisan, including the 4-methylene cyclohexane-1-carbonyl moietyshown above.

The immunoconjugate of the preferred embodiments of the presentinvention is obtained from the reaction of the activatedchemotherapeutic moiety with an antibody using methods well known in theart. The chemotherapeutic moiety may be attached to the mAb through thethiol-reactive group after reduction of the mAb inter-chain disulfidebonds. This approach generates an average of eight free thiol groups permolecule of antibody, and does so in a reproducible manner at thelimiting levels of thiol used in the reduction reaction.

For conjugating to lysine groups of an antibody, the antibody is firstderivatized to contain a thiol-reactive group, with the chemotherapeuticmoiety containing a thiol group. Methods for introducing thiol groups onto antibodies by modifications of mAb's lysine groups are well known inthe art (Govindan et al. Bioconjugate Chemistry, 7:290-297, 1996; Wongin Chemistry of protein conjugation and cross-linking, CRC Press, Inc.,Boca Raton, Fla. (1991), pp 20-22). In this way, the bifunctionalchemotherapeutic moiety is conjugated to antibody fragments andsubfragments including single-chain constructs. Conversely, thechemotherapeutic moiety contains a thiol-reactive group which reactswith the thiol(s) of a reduced mAb.

The targeting moiety is preferably an antibody (including fully human,non-human, humanized, or chimeric antibodies) or an antibody fragment(including enzymatically or recombinantly produced fragments) andbinding proteins incorporating sequences from antibodies or antibodyfragments. The antibodies, fragments, and binding proteins may bemultivalent and multispecific or multivalent and monospecific as definedabove.

In a preferred embodiment of the present invention, antibodies, such asmAbs, are used that recognize or bind to markers or tumor-associatedantigens that are expressed at high levels on target cells and that areexpressed predominantly or only on diseased cells versus normal tissues,and antibodies that internalize rapidly. Antibodies useful within thescope of the present invention include mAbs with properties as describedabove (and show distinguishing properties of different levels ofinternalization into cells and microorganisms), and contemplate the useof, but are not limited to, in cancer, the following mAbs: LL1(anti-CD74), LL2 (anti-CD22), RS7 (anti-epithelialglycoprotein-1(EGP-1)), PAM-4 and KC4 (both anti-MUC1), MN-14(anti-carcinoembryonic antigen (CEA, also known as CD66e), Mu-9(anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein),TAG-72 (e.g., CC49), Tn, J591 (anti-PSMA (prostate-specific membraneantigen)), G250 (an anti-carbonic anhydrase IX mAb) and L243(anti-HLA-DR). Other useful antigens that may be targeted using theseconjugates include HER-2/neu, BrE3, CD19, CD20 (e.g., C2B8, hA20, 1F5Mabs) CD21, CD23, CD80, alpha-fetoprotein (AFP), VEGF, EGF receptor,P1GF, MUC1, MUC2, MUC3, MUC4, PSMA, gangliosides, HCG, EGP-2 (e.g.,17-IA), CD37, HLA-DR, CD30, Ia, A3, A33, Ep-CAM, KS-1, Le(y), S100, PSA(prostate-specific antigen), tenascin, folate receptor,Thomas-Friedenreich antigens, tumor necrosis antigens, tumorangiogenesis antigens, Ga 733, IL-2, IL-6, T101, MAGE, antigen to whichL243 binds, CD66 antigens, i.e. CD66a-d or a combination thereof. TheCD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens are expressed mainly in granulocytes, normal epithelial cellsof the digestive tract anc tumor cells of various tissues. A number ofthe aforementioned antigens are disclosed in U.S. ProvisionalApplication Ser. No. 60/426,379, entitled “Use of Multi-specific,Non-covalent Complexes for Targeted Delivery of Therapeutics,” filedNov. 15, 2002.

In another preferred embodiment of the present invention, antibodies areused that internalize rapidly and are then re-expressed, processed andpresented on cell surfaces, enabling continual uptake and accretion ofcirculating immunoconjugate by the cell. An example of a most-preferredantibody/antigen pair is LL1, an anti-CD74 mAb (invariant chain, classII-specific chaperone, Ii). The CD74 antigen is highly expressed onB-cell lymphomas, certain T-cell lymphomas, melanomas and certain othercancers (Ong et al., Immunology 98:296-302 (1999)), as well as certainautoimmune diseases.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, melanoma andmultiple myeloma. Continual expression of the CD74 antigen for shortperiods of time on the surface of target cells, followed byinternalization of the antigen, and re-expression of the antigen,enables the targeting LL1 antibody to be internalized along with anychemotherapeutic moiety it carries as a “payload.” This allows a high,and therapeutic, concentration of LL1-chemotherapeutic drugimmunoconjugate to be accumulated inside such cells. InternalizedLL1-chemotherapeutic drug immunoconjugates are cycled through lysosomesand endosomes, and the chemotherapeutic moiety is released in an activeform within the target cells.

In another aspect, the invention relates to a method of treating asubject, comprising administering a therapeutically effective amount ofa therapeutic conjugate of the preferred embodiments of the presentinvention to a subject. Diseases that may be treated the therapeuticconjugates of the preferred embodiments of the present inventioninclude, but are not limited to B-cell malignancies (e.g., non-Hodgkinslymphoma and chronic lymphocytic leukemia using, for example LL2 mAb;see U.S. Pat. No. 6,183,744), adenocarcinomas of endodermally-deriveddigestive system epithelia, cancers such as breast cancer and non-smallcell lung cancer, and other carcinomas, sarcomas, glial tumors, myeloidleukemias, etc. In particular, antibodies against an antigen, e.g., anoncofetal antigen, produced by or associated with a malignant solidtumor or hematopoietic neoplasm, e.g., a gastrointestinal, lung, breast,prostate, ovarian, testicular, brain or lymphatic tumor, a sarcoma or amelanoma, are advantageously used.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals.The term subject also includes rodents (e.g., mice, rats, and guineapigs). It is not intended that the term be limited to a particular ageor sex. Thus, adult and newborn subjects, as well as fetuses, whethermale or female, are encompassed by the term.

In another preferred embodiment, the therapeutic conjugates comprisingthe Mu-9 mAb of the preferred embodiments of the present invention canbe used to treat colorectal, as well as pancreatic and ovarian cancersas disclosed in U.S. application Ser. No. 10/116,116, filed Apr. 5, 2002and by Gold et al. (Cancer Res. 50: 6405 (1990), and references citedtherein). In addition, the therapeutic conjugates comprising the PAM-4mAb of the preferred embodiments of the present invention can be used totreat pancreatic cancer, as disclosed in U.S. Provisional ApplicationSer. No. 60/388,314, filed Jun. 14, 2002.

In another preferred embodiment, the therapeutic conjugates comprisingthe RS-7 mAb of the preferred embodiments can be used to treatcarcinomas such as carcinomas of the lung, stomach, urinary bladder,breast, ovary, uterus, and prostate, as disclosed in U.S. ProvisionalApplication Ser. No. 60/360,229, filed Mar. 1, 2002 and by Stein et al.(Cancer Res. 50: 1330 (1990) and Antibody Immunoconj. Radiopharm. 4: 703(1991)).

In another preferred embodiment, the therapeutic conjugates comprisingthe anti-AFP mAb of the preferred embodiments can be used to treathepatocellular carcinoma, germ cell tumors, and other AFP-producingtumors using humanized, chimeric and human antibody forms, as disclosedin U.S. Provisional Application Ser. No. 60/399,707, filed Aug. 1, 2002.

In another preferred embodiment, the therapeutic conjugates comprisinganti-tenascin antibodies can be used to treat hematopoietic and solidtumors and conjugates comprising antibodies to Le(y) can be used totreat solid tumors.

In another preferred embodiment, diseases that may be treated using thetherapeutic conjugates of the preferred embodiments of the presentinvention include, but are not limited to immune dysregulation diseaseand related autoimmune diseases, including Class III autoimmune diseasessuch as immune-mediated thrombocytopenias, such as acute idiopathicthrombocytopenic purpura and chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sjögren's syndrome, multiple sclerosis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, sarcoidosis, ulcerativecolitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa,ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,rheumatoid arthritis, polymyositis/dermatomyositis, polychondritis,pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis, as disclosed in U.S.Provisional Application Ser. No. 60/360,259, filed Mar. 1, 2002. Typicalantibodies useful in these diseases include, but are not limited to,those reactive with HLA-DR antigens, B-cell antigens (e.g., CD19, CD20,CD21, CD22, CD23, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22,CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, B7, MUC1, Ia, HM1.24, and HLA-DR). Since many of these autoimmunediseases are affected by autoantibodies made by aberrant B-cellpopulations, depletion of these B-cells by therapeutic conjugatesinvolving such antibodies bound with the drugs used in this invention,is a preferred method of autoimmune disease therapy, especially whenB-cell antibodies are combined, in certain circumstances, with HLA-DRantibodies and/or T-cell antibodies (including those which target IL-2as an antigen, such as anti-TAC antibody).

In another preferred embodiment, the therapeutic conjugates of thepreferred embodiments can be used against pathogens, since antibodiesagainst pathogens are known. For example, antibodies and antibodyfragments which specifically bind markers produced by or associated withinfectious lesions, including viral, bacterial, fungal and parasiticinfections, for example caused by pathogens such as bacteria,rickettsia, mycoplasma, protozoa, fungi, and viruses, and antigens andproducts associated with such microorganisms have been disclosed, interalia, in Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg U.S. Pat.Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,818,709and 4,624,846. In a preferred embodiment, the pathogens are selectedfrom the group consisting of Streptococcus agalactiae, Legionellapneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhosae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzaeB, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, mycobacterium tuberculosis,rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I,herpes simplex virus II, human serum parvo-like virus, respiratorysyncytial virus, varicella-zoster virus, hepatitis B virus, measlesvirus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus,murine leukemia virus, mumps virus, vesicular stomatitis virus, sindbisvirus, lymphocytic choriomeningitis virus, wart virus, blue tonguevirus, Sendai virus, feline leukemia virus, reo virus, polio virus,simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus,Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosomarangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosomabrucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis,Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinellaspiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taeniasaginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasmaarthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasmalaidlawii, M. salivarium and M. pneumoniae, as disclosed in U.S. Pat.No. 6,440,416.

In one embodiment, antibiotics are delivered as chemoconjugatesincorporating siderophore components for use against infection withGram-negative bacterial pathogens. Siderophores are iron-chelating hiscatecholates of di- or triamines, and are used as delivery vehicles. Theiron chelate of siderophores are recognized by specific receptors on thebacterial membrane, thus leading to active transport inside thebacterila cell (Heinisch et al. J. Medicinal Chemistry, 45: 3032-3040,2002). Within the context of the present invention, a peptide isassembled with protected lysine as the N-terminus. The carboxyl end ofthe peptide, liberated from the resin support, is then activated, andcoupled to amine-containing antibiotic such as penicillin N. Theprotecting groups at the N-terminus are cleaved, and the amino groups ofthe N-terminal lysine are then conjugated to 2,3-diacetoxybenzoylchloride to ultimately furnish the required bis catecholate-appendedpenicillin conjugate. The diacetyl derivative of the catechol moiety isalso effective as siderophore component (Heinisch et al. supra).

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. For veterinary uses, the same-species IgG would likely be the mosteffective vector, although cross-species IgGs would remain useful. Samespecies IgG molecules as delivery agents are mostly preferred tominimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Targeting an internalizing antigen, antibodies such as hLL1and hLL2 rapidly internalize after binding to target cells, which meansthat the chemotherapeutic drug being carried is rapidly internalizedinto cells. However, antibodies that have slower rates ofinternalization can also be used to effect selective therapy with thisinvention.

In a preferred embodiment of this invention, a more effectiveincorporation into cells and pathogens can be accomplished by usingmultivalent, multispecific or multivalent, monospecific antibodies.Multivalent means the use of several binding arms against the same ordifferent antigen or epitope expressed on the cells, whereasmultispecific antibodies involve the use of multiple binding arms totarget at least two different antigens or epitopes contained on thetargeted cell or pathogen. Examples of such bivalent and bispecificantibodies are found in U.S. patent applications 60/399,707, filed Aug.1, 2002; 60/360,229, filed Mar. 1, 2002; 60/388,314, filed Jun. 14,2002; and Ser. No. 10/116,116, filed Apr. 5, 2002, all of which areincorporated by reference herein.

In a preferred embodiment of the present invention, camptothecin (CPT)and its derivatives are preferred chemotherapeutic moieties, althoughthe invention is not so limited. Other chemotherapeutic moieties thatare within the scope of the invention are taxanes (e.g, baccatin III,taxol), epothilones, anthracycline drugs (doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), and 2-pyrrolinodoxorubicin (2-PDOX); see Priebe W(ed.), ACS symposium series 574, published by American Chemical Society,Washington D.C., 1995. (332 pp) and Nagy et al., Proc. Natl. Acad. Sci.USA 93:2464-2469, 1996), benzoquinoid ansamycins exemplified bygeldanamycin (DeBoer et al., Journal of Antibiotics 23:442-447, 1970;Neckers et al., Invest. New Drugs 17:361-373, 1999), and the like.Preferably, in the immunoconjugates of the preferred embodiments of thepresent invention, the targeting moiety links to at least onechemotherapeutic moiety; preferably 1 to about 5 chemotherapeuticmoieties; most preferably about 7 to about 12 chemotherapeutic moieties.

CPT derivatives are a class of potent antitumor agents. Irinotecan (alsoreferred to as CPT-11) and topotecan are CPT analogs which are approvedcancer therapeutics (Iyer and Ratain, Cancer Chemother. Pharmacol. 42:S31-S43 (1998)). CPTs act by inhibiting topoisomerase I enzyme bystabilizing topoisomerase I-DNA complex (Liu, et al. in TheCamptothecins: Unfolding Their Anticancer Potential, Liehr J. G.,Giovanella, B. C. and Verschraegen (eds), NY Acad. Sci., NY (2000), pp1-10).

CPTs present a set of caveats in the preparation of immunoconjugates.One caveat is the insolubility of most CPT derivatives in aqueousbuffers. Secondly, CPTs provide very few options for structuralmodification for conjugating to macromolecules. For instance, CPT itselfcontains only a tertiary hydroxyl group in ring-E. The hydroxylfunctional group in the case of CPT must be coupled to a linker suitablefor subsequent protein conjugation. Thirdly the lability of theδ-lactone moiety of the E-ring of their structures, under physiologicalconditions, results in greatly reduced antitumor potency of theseproducts. Therefore, the conjugation protocol is performed such that itis carried out at a pH of 7 or lower to avoid the lactone ring opening.Typically conjugation of a bifunctional CPT possessing an amine-reactivegroup such as an active ester would require a pH of 8 or greater.Fourth, an intracellularly-cleavable moiety is to be incorporated in thelinker/spacer connecting the CPTs and the antibodies.

The problem of δ-lactone opening under physiological conditions has beenpreviously addressed. One approach has been to acylate the C-20 hydroxylgroup with an amino acid, and couple the α-amino group of the amino acidto poly-L-glutamic acid. This approach relies on the passive diffusionof a polymeric molecule into tumor sites. This glycine conjugation hasalso been reported as a method of making water-soluble derivative of CPT(U.S. Pat. No. 4,943,579) and in the preparation of a PEG-derivatizationof CPT (Greenwald, et al. J. Med. Chem. 39: 1938-1940 (1996). In thelatter case, the approach has been devised in the context of developingwater-soluble and long acting forms of CPT, whereby CPT's in vivohalf-life is enhanced, and the drug is gradually released from itsconjugate while in circulation in vivo.

In a preferred embodiment, the present invention devises methods forpreparing immunoconjugates of CPTs, taking into consideration the fourcaveats described above. The preferred embodiments of the presentinvention address the caveats in the following ways. First, thewater-solubility of CPT is increased by placing an aminopolycarboxylatebetween the chemotherapeutic moiety (i.e., CPT) and the antibody. In apreferred embodiment the aminopolycarboxylate is selected from the groupconsisting of DTPA (diethylenetriaminepentaacetic acid), NOTA(1,4,7-triazacyclononane-N,N′,N″-triacetic acid), DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid), and TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid).

In a preferred embodiment, the aminopolycarboxylate is DTPA. In aparticularly preferred embodiment, the DTPA is attached to thechemotherapeutic moiety by way of a reagent bearing the same.Preferably, said reagent bearing an aminopolycarboxylate is:

wherein “Trt” is a trityl group and the R groups on the carboxylates areeither hydrogen or alkyl (wherein alkyl means a straight or branchedchain C₁-C₆-alkyl group). The ordinary skilled artisan would be able todevelop other reagents bearing an aminopolycarboxylate that would beuseful in the practice of the preferred embodiments of the presentinvention. The reagent bearing a hapten can be attached to thechemotherapeutic moiety via the carboxylic acid of the thiourea moietyby methods well know in the art. One such method well know in the art isdescribed in Example 8. Where R is alkyl, the esters are deprotected toultimately obtain the corresponding DTPA derivative.

The reagent bearing an aminopolycarboxylate may be attached to thechemotherapeutic moiety directly or by way of an amino acid spacer. In apreferred embodiment, the reagent bearing an aminopolycarboxylate isattached to the chemotherapeutic moiety via a glycine spacer, and morepreferably via valine spacer. The use of more hindered valinate esterhas been shown to provide a hydrolytically stable moiety (Lerchen H-G etal., J. Med. Chem. 44:4186-4195 (2001)). The derivatization of C-20hydroxyl as an ester can also involve a dipeptide or a polypeptide. Whenthe chemotherapeutic moiety is 10-hydroxy CPT, the glycyl spacer isattached either at the A-ring hydroxyl group or at the C-20 hydroxylgroup as shown in Example 7, below; most preferably, the glycyl or otherspacers mentioned above is attached at the C-20 hydroxyl group.

In a preferred embodiment, the water-solubility of the chemotherapeuticmoiety is increased via the incorporation of the following moiety (A) ata hydroxyl group on the chemotherapeutic moiety:

wherein “PEG” denotes polyethylene glycol. This moiety not onlyincreases the water solubility of the chemotherapeutic moiety, but italso incorporates a thiol reactive group. In the case of 10-hydroxy CPT,the aforementioned moiety may be attached at the A-ring hydroxyl groupor at the C-20 hydroxyl group.

For attachment to the C-20 hydroxyl, the C-10 hydroxyl is firstprotected. The C-20 hydroxyl is then converted to, for example, theglycinate ester. At this point, the C-10 hydroxyl may be deprotected.The free amino group of the C-20 glycinate ester can then be suitablyelaborated to contain, for example, an intracellularly cleavable‘Phe-Lys’ dipeptide linkage which is recognized and cleaved byintracellular Cathepsin B enzyme. The PEG-containing moiety shown aboveis then installed at the free amino group at the end of the C-20glycinate-Phe-Lys dipeptide.

Second, the δ-lactone is stabilized via derivatization of the C-20hydroxyl group with an amino acid radical. The use of an amino acidlinker/spacer at the C-20 hydroxyl of CPT is advantageous for furtherelaboration of the amine with a thiol-reactive moiety. Moreover, theamino acid linker/spacer acts as an intracellularly-cleavable moiety. Atthis point, the immunoconjugate effectively becomes anintracellularly-activated prodrug. That is, the release of thechemotherapeutic moiety from the immunoconjugate would depend upon thebinding of the immunoconjugate to tumor-specific antigens, subsequentinternalization, routing to lysosomal compartment, and finallyprocessing of the conjugate by lysosomal enzymes resulting in therelease of the drug.

Finally, the conjugation protocol is based on a thiol-maleimide or athiol-vinylsulfone reaction which is facile at neutral or acidic pH.This obviates the need for higher pH conditions for conjugations as, forinstance, would be necessitated when using active esters.

In another aspect, the present invention relates to a modular approachfor introducing a water-solubilizing moiety and a thiol-reactive moiety(e.g., maleimide groups) into the immunoconjugates of the preferredembodiments of the present invention. The water-solubilizing moiety andthe thiol-reactive moiety are conveniently introduced onto a templatesuch as: MCC-Gly-Lys-Lys(R′)—NH₂, wherein MCC is4-maleimidomethyl-cyclohexyl-1-carbonyl (the maleimide derivative ofchoice derived from SMCC), and wherein R′ is a water solubilizingentity, such as an aminopolycarboxylate (e.g., DTPA) or a derivative ofpiperazine. The derivatization of CPT or its analog as an ester at theC-20 hydroxyl, through the use of an amino acid or a dipeptide asenumerated in the embodiments described already, results in an aminogroup which is available for activation. The amino group can beconverted to an isothiocyanato group by methods well known in the art;the latter is linked to the side chain amino group of the internallysine moiety of the peptide template described above. In this way, awater-solubilizing group and an antibody-reactive group are added in asingle step. Moreover, by modifying the peptide template to contain morethan one central lysine or other side-chain-amine-containing amino acid,such as arginine, multiple chemotherapeutic drug moieties can be added.

It is contemplated that the above-mentioned approach can be useful inintroducing 1 to 5, more preferably 1 to 2 chemotherapeutic drugmoieties onto the linker.

In yet another preferred embodiment, a pH-sensitiveintracellularly-cleavable moiety is used as a linker binding thetargeting moiety to the chemotherapeutic moiety. An advantage with thisapproach is that intratumoral release of the chemotherapeutic moietywill not depend upon the extent of expression of specific enzymes forthe breakage of the bonds of the intracellularly-cleavable moiety thatlinks the targeting moiety to the chemotherapeutic moiety.

For example, the 20-hydroxyl group of CPT can be attached under acidcatalysis to a pH-sensitive intracellularly-cleavable moiety comprisinga dihydropyran moiety, a tetrahydrofuran moiety or an orthoester moietywhich, in turn, comprises a thiol-reactive moiety (e.g., maleimide), andwater solubilizing group (e.g., piperazine). See Chart 1. pH-sensitiveintracellularly-cleavable moieties comprising a dihydropyran,tetrahydrofuran or an orthoester moiety all comprise an ether bond thatis susceptible to cleavage under the acidic pH of intracellularcompartments.

A linker with structure-1 in the chart below is used. In this example,the central piperazine portion will allow for water-solubility. Thesubstituted dihydropyran at the ‘left end’ of the molecule is forcoupling to the hydroxyl group(s) in CPT derivatives, while themaleimide at the ‘right end’ of the molecule is a prototypicalthiol-reactive group for conjugating to disulfide-reduced antibody.Herein, ‘CPT’ is used as a general term, and can be CPT, 10-hydroxy-CPT,or SN-38, as shown in the Chart. In the case of 10-hydroxy-CPT andSN-38, derivatizations can be envisaged at both 10-hydroxy and20-hydroxy positions. The bonding of CPT to the cross-linker is via atetrahydropyran moiety. This bond is slowly cleaved at pH 4-6, butstable above pH 6 (Greene T W, Wuts P G M: Protective groups in organicsynthesis, second edition; John Wiley & Sons: New York, 1991; pp413-416.) This approach takes advantage of the acidic nature ofintracellular compartment; generally about 5. (Poznansky M J and JulianoR L, Pharmacol Rev 36:277-336, 1984.) This acidic pH in intracellularcompartments causes the release the drug intact from the antibody.

As mentioned above, tetrahydrofuranyl moieties are contemplated asintegral parts of pH-sensitive intracellularly-cleavable moieties.(Greene T W, Wuts P G M: Protective groups in organic synthesis, secondedition; John Wiley & Sons: New York, 1991; pp 267-269.) When thepH-sensitive intracellularly cleavable moiety comprises atetrahydrofuranyl moiety, the cleavage of the CPTC20-oxygen-tetrahydrofuran bond cleaves faster than when atetrahydropyranyl moiety is used.

Suitable routes of administration of the immunoconjugates of thepreferred embodiments of the present invention include, withoutlimitation, oral, parenteral, rectal, transmucosal, intestinaladministration, intramuscular, subcutaneous, intramedullary,intrathecal, direct intraventricular, intravenous, intravitreal,intraperitoneal, intranasal, or intraocular injections. The preferredroutes of administration are parenteral. Alternatively, one mayadminister the compound in a local rather than systemic manner, forexample, via injection of the compound directly into a solid tumor.

The present invention is illustrated by the following examples, withoutlimiting the scope of the invention.

EXAMPLES Example-1 Preparation of C-20-O—(N-‘MCC’)glycinate derivativeof 10-hydroxycamptothecin, where MCC is4-(N-maleimidomethyl)-cyclohexane-1-carbonyl residue

10-Hydroxycamptothecin (0.2081 g; 0.57 mmol) was dissolved in 20 mL ofanhydrous dimethyl formamide (DMF) and 0.22 mL of diisopropylethylamine(DIEA). To this was added 0.1248 g (0.619 mmol) of 4-nitrophenylchloroformate, and the reaction mixture was stirred under argon, at theroom temperature, for 4 h. A solution of 4-piperidinopiperidine (0.2403g) in 4 mL of DMF was added in one lot, and the mixture was stirred foranother 2 h. Solvent removal furnished the crude product, which waspurified by flash chromatography on silica gel (230-400 mesh) usingmethanol-dichloromethane gradient elution to obtain 52.8 mg of theintermediate 2. Electrospray mass spectrum showed a clean peak at m/e559 (M+H) in the positive ion mode, and peaks at m/e 557 (M−H) and 593(M+Cl) in the negative ion mode. The intermediate 2 (25 mg) was reactedwith BOC-glycine (8.9 mg), N,N-dimethylaminopyridine (DMAP) (2 mg) anddicyclohexylcarbodiimide (DIC) (0.058 mL of 1 M solution indichloromethane) in 10 mL of dichloromethane (DCM) for 18 h (roomtemperature, under argon). The reaction mixture was worked up andpurified by flash chromatography to obtain C-20-O-(BOC)glycinate in ˜50%yield. This product was reacted with a 10% DCM solution oftrifluoroacetic acid (TFA), for 1-to-2 h, to obtain 20-O-glycinatederivative. The latter was derivatized with 1.2 molar equivalent ofsuccinimidylmaleimidomethylcyclohexanecarboxylate (SMCC) and 1.5equivalent of DIEA in DMF for ˜3 h (room temperature, under argon).Solvent removal and flash chromatography resulted in the recovery of 7.8mg of pure 3 as gummy solid. 3: Analytical HPLC (C₁₈ column, gradientelution using solution A changing to solution ‘B’ in 10 minutes at 3mL/min, then maintained at 100% ‘B’ for 5 min.; ‘A’: 0.1% aq. TFA; ‘B’:90% CH₃CN/0.1% TFA) showed single peak 9.48 min (absorbance at 360 nm).Electrospray mass spectrum showed mass peak at m/e 835 (M+H).

Example-2 Preparation of C-20-O—(N-‘MCC’)glycinate derivative ofirinotecan (CPT-11), where MCC is4-(N-maleimidomethyl)-cyclohexane-1-carbonyl residue

Starting from CPT-11 (4), the product 5 was prepared in a 3-stepprocedure by reacting with BOC-glycine (purified product showed M+H atm/e 744 in electrospray mass spectrum), deprotection with TFA (M+H peakat m/e 644 in mass spectrum), and coupling to SMCC, as in Example-1.Product purifications were carried out as in Example-1. The finalproduct 5 showed an intense peak at m/e 863 in the positive ion modeelectrospray mass spectrum, and a clean peak in analytical HPLC at 9.69min. Pure product 5 was obtained as a white solid, and was produced in25 mg amount.

Example-3 Preparation of 10-piperazinocarbonyloxy camptothecin (6) andits coupling to a heterobifunctional poly(ethyleneglycol),maleimide-PEG-NHS

The preparation of the intermediate 6 in the Scheme-3 above was achievedalong the lines of the preparation of 2 in Scheme-1 by using piperazinein place of piperidinopiperidine. After flash chromatographicpurification, 6 was obtained in 43.3% yield (orange solid). Electrospraymass spectrum showed M+H at m/e 477 and M−H (negative ion mode) at m/e475. For the preparation of 7, the precursor 6 was mixed with 0.5 molarequivalent of poly(ethylene glycol-α-N-hydroxysuccinimidyl propionate,β-maleimide, MW 3400 (obtained from Shearwater Polymers, Huntsville,Ala.) in DMF, and incubated for 30 min. The reaction mixture was used assuch without purification. The molar content of the activated CPT wasconsidered equal to that of the limiting PEG reagent used.

Example-4 General Procedure for Conjugating CPT Derivatives 3 or 5 or 7with Monoclonal Antibodies

The antibody was treated with a 20-to-40-fold molar excess ofdithiothreitol and ethylene diamine tetraacetic acid (EDTA) (˜5 mM finalconc.) at pH 7.4, flushed with argon, and incubated for 45 min. at 37°C. The cooled contents were then purified on two successive 3-mL columnsof Sephadex G50/80 in 50 mM sodium acetate-150 mM sodium chloride pH 5.3(“ABS buffer”) under centrifuged elution conditions (centrifuged SEC).The eluate was analyzed for protein concentration using absorbance at280 nm, and thiol content by Ellman's assay. The reduced antibodiescontained 7-to-9 thiol groups. The reduced antibody was reacted with a20-fold molar excess of the CPT derivative in DMF such that the finalconcentration of DMF in the conjugation mixture was ≦5% v/v, andincubated on ice (˜4° C.) for 20-to-30 min. Two successivecentrifuged-SEC purifications on Sephadex G50/80 in ABS buffer, followedby passing the eluate through a hydrophobic column to removenon-covalently bound CPT derivative, furnished the final conjugate. Inthe case of conjugates using 7, additional purification byultrafiltration on a 30K MWCO centrifugal filter proved necessary. Inall the conjugate preparations, the reaction mixtures as well as thepurified conjugates were clear solutions, indicating that thewater-soluble nature of CPT derivatives used. Analysis by SE-HPLC wascarried out on an analytical SEC250 column, fitted with a guard columnand an in-line absorbance detector, using 0.2 M sodium phosphate pH 6.8as eluent and 1 mL/min as the flow rate. The product was also analyzedby spectrophotometric absorbance scan in the 260-540 nm region.Absorbance at 360 nm was correlated with that obtained with a CPT-11standard to determine the CPT concentration in the conjugate. Absorbanceat 280 nm, corrected for spillover from CPT derivative, was used tocalculate the antibody concentration. This way, the CPTderivative-to-IgG molar ratio was determined. The product was mixed with10% v/v of 1 M sucrose, aliquoted in 1 mg and 0.1 mg lots, andlyophilized. The lyophilized preparations were stored in freezer afterargon flush.

Example-5 MAb Conjugates of a Derivative of 10-Hydroxy CPT, 3

The conjugates were prepared as described in Example-4. The followingare the data pertaining to various conjugates.

Murine LL2 (anti-CD-22 MAb) conjugate of 3: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showedpredominantly a single peak eluting at the retention time of unmodifiedLL2, the latter analyzed under the same conditions with detection set at280 nm. Aggregate content: 1.6%. Molar substitution of 3 in theconjugate (i.e. 3-to-IgG ratio) was found to be 8.3 by absorbance scan,and 6 by MALDI mass spectral analysis.Murine LL1 (anti-CD-74 MAb) conjugate of 3: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showedpredominantly a single peak eluting at the retention time of unmodifiedLL1, the latter analyzed under the same conditions with detection set at280 nm. Aggregate content: 2.5%. Molar substitution of 3 in theconjugate (i.e. 3-to-IgG ratio) was found to be 7.9 by absorbance scan.Murine RS7 (anti-EGP-1 MAb) conjugate of 3: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showedpredominantly a single peak eluting at the retention time of unmodifiedRS7, the latter analyzed under the same conditions with detection set at280 nm. Aggregate content: 1.4%. Molar substitution of 3 in theconjugate (i.e. 3-to-IgG ratio) was found to be 6.9 by absorbance scan.Humanized LL2 (anti-CD-22 MAb) conjugate of 3: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showedpredominantly a single peak eluting at the retention time of unmodifiedhLL2, the latter analyzed under the same conditions with detection setat 280 nm. Aggregate content: 0%. Molar substitution of 3 in theconjugate (i.e. 3-to-IgG ratio) was found to be 7.1 by absorbance scan.Humanized AFP31 (anti-AFP MAb) conjugate of 3: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showedpredominantly a single peak eluting at the retention time of unmodifiedhAFP31, the latter analyzed under the same conditions with detection setat 280 nm. Aggregate content: 0%. Molar substitution of 3 in theconjugate (i.e. 3-to-IgG ratio) was found to be 8.0 by absorbance scan.

Example-6 MAb Conjugates of a Derivative of CPT-11, 5

The conjugates were prepared as described in Example-4. The followingare the data pertaining to various conjugates.

Murine LL2 (anti-CD-22 MAb) conjugate of 5: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showedpredominantly a single peak eluting at the retention time of unmodifiedLL2, the latter analyzed under the same conditions with detection set at280 nm. Aggregate content: 2.2%. Molar substitution of 5 in theconjugate (i.e. 5-to-IgG ratio) was found to be 9.7 by absorbance scan.Murine LL1 (anti-CD-74 MAb) conjugate of 5: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showeda peak (91.2%) eluting at the retention time of unmodified LL1, thelatter analyzed under the same conditions with detection set at 280 nm.Aggregate content: 8.8%. Molar substitution of 5 in the conjugate (i.e.5-to-IgG ratio) was found to be 10.9 by absorbance scan.Murine MN-14 (anti-CEA MAb) conjugate of 5: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showeda peak eluting at the retention time of unmodified MN-14, the latteranalyzed under the same conditions with detection set at 280 nm.Aggregate content: 9.3%. Molar substitution of 5 in the conjugate (i.e.5-to-IgG ratio) was found to be 10.0 by absorbance scan.Humanized LL2 (anti-CD-22 MAb) conjugate of 5: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showedpredominantly a single peak eluting at the retention time of unmodifiedhLL2, the latter analyzed under the same conditions with detection setat 280 nm. Aggregate content: 0%. Molar substitution of 5 in theconjugate (i.e. 5-to-IgG ratio) was found to be 8.4 by absorbance scan.

Example-7 MAb Conjugates of a PEG-Derivative of 10-Hydroxy CPT, 7

The conjugates were prepared as described in Example-4. The followingare the data pertaining to various conjugates.

Murine LL2 (anti-CD-22 MAb) conjugate of 7: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showeda peak (83%) eluting near the retention time of unmodified LL2, alongwith aggregate content of 16.3%. Molar substitution of 7 in theconjugate (i.e. 7-to-IgG ratio) was found to be 8.0 by absorbance scan.Murine LL1 (anti-CD-74 MAb) conjugate of 7: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showeda peak (94%) eluting near the retention time of unmodified LL1, alongwith aggregate content of 6.0%. Molar substitution of 7 in the conjugate(i.e. 7-to-IgG ratio) was found to be 8.1 by absorbance scan.Murine RS7 (anti-EGP-1 MAb) conjugate of 7: SE-HPLC analysis, withabsorbance detection set at the CPT absorbance maximum of 360 nm, showeda peak eluting near the retention time of unmodified RS7, along withaggregate content of 4.5%. Molar substitution of 7 in the conjugate(i.e. 7-to-IgG ratio) was found to be 8.0 by absorbance scan.

Example-8 A General Method for the Preparation ofAminopolycarboxylate-Appended Bifunctional CPT(s), Suitable forConjugation to Lysine or Thiol Group on an Antibody

The general approach is shown in Scheme-4. Briefly, CPT and10-hydroxy-CPT were converted to the corresponding C-20-O-glycinate bythe method of Singer (supra). In a parallel synthesis,isothiocyanatobenzyl DTPA penta t-butyl ester was generated from thecorresponding amine precursor, and then coupled to S-trityl cysteine.Flash chromatographic purification gave the intermediate 11 (massspectrum: M+Na m/e 1207; M−H: 1183).

Intermediate 11 was then coupled to the CPT derivative 9. TFA-mediateddeprotection of t-butyl groups of carboxylates and S-trityl group, usingscavengers to capture t-butyl cation, then furnished the DTPA-appendedbifunctional CPT derivative (R═H or OH), suitable for conjugating tomaleimide groups appended to IgG.

The thiol group is also derivatized with excess divinylsulfone toproduce an intermediate possessing a vinylsulfone group, which issuitable for conjugating to thiol groups of disulfide-reducedantibodies.

Intermediate 13 is another example of an aminopolycarboxylate which canbe used in place of isothiocyanatobenzyl DTPA in these transformations.

Example-9 Method for the Preparation of SN-38 Conjugates

SN-38 is the active drug which is metabolically converted, in vivo, fromthe prodrug CPT-11 (Liehr, Giovanella, Verschraegen (eds) (supra). SN-38is the active chemotherapeutic drug which is 3 orders of magnitude morepotent than CPT-11 in inhibiting topoisomerase I activity. BifunctionalSN-38, suitable for antibody conjugation is simply prepared as follows:[CPT-11]-20-O-valinate, with its valine N-terminus protected as BOC, isselectively cleaved at its C-10-carbamate using one equivalent of a basesuch as hydroxide. “Anhydrous hydroxide”, such as that derived from 2equivalents of potassium t-butoxide and one equivalent of water (Gassmanand Schenk, J. Org. Chem., 42:918-920 (1977)), is advantageously usedfor this purpose. The valinate ester of CPT has been shown to be stableto 1 equivalent of 1N aq. sodium hydroxide for at least 1 hour. Thisway, the C-10-carbamate is selectively cleaved. The amino group of thevaline moiety is then converted to isothiocyanate, and coupled to thepeptide template MCC-Gly-Lys-Lys(R′)—NH₂. Alternatively, SN-38 itself isderivatized by first protecting the C10-hydroxyl group as an acidsensitive derivative, such as methoxytrityl derivative. The C20-hydroxylgroup is then converted to the valinate ester. At this point, themethoxytrityl group at C-10 position is removed. A different optionwould be to first prepare the 20-valinate ester (or any other ester)derivative of SN-38 via protection of C-10 hydroxyl as the corresponding‘BOC’ ester, derivatization of 20-hydroxyl group, and deprotection. Thisis then followed by treatment with trimethylsilyl chloride to derivativeboth C-10 hydroxyl and the amino group of the ester at C-20, reactingwith amine-reactive heterobifunctional cross-linker, such asmaleimide-PEG-NHS, and deprotecting trimethylsilyl group at C-10position.

The above approach is not limited to CPT derivatives, and is applicableto other hydrophobic drugs such as paclitaxel, geldanamycin, and thelike.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated by referencein their entirety.

The CDRs of the heavy chain variable region of the humanized anti-AFPMab comprises CDR1 comprising an amino acid sequence of SYVIH (SEQ IDNO:1); CDR2 comprising an amino acid sequence of YIHPYNGGTKYNEKFKG (SEQID NO:2) and CDR3 comprising an amino acid sequence of SGGGDPFAY (SEQ IDNO:3) and the CDRs of the light chain variable region of the humanizedanti-AFP Mab comprises CDR1 comprising an amino acid sequence ofKASQDINKYIG (SEQ ID NO:4), CDR2 comprising an amino acid sequence ofYTSALLP (SEQ ID NO:5) and CDR3 comprising an amino acid sequence ofLQYDDLWT (SEQ ID NO:6).

The CDRs of the light chain variable region of the humanized RS7comprises CDR1 comprising an amino acid sequence of KASQDVSIAVA (SEQ IDNO:7), SASYRYT (SEQ ID NO:8) and QQHYITPLT (SEQ ID NO:9) and the CDRs ofthe heavy chain variable region of the humanized anti-CD20 MAb comprisessequence comprising CDR1 comprising an amino acid sequence of NYGMN (SEQID NO:10), CDR2 comprising an amino acid sequence of WINTYTGEPTYIDDFKG(SEQ ID NO:11) and CDR3 comprising an amino acid sequence ofGGFGSSYWYFDV (SEQ ID NO:12).

The light chain variable region of the humanized LL1 mAb comprises CDRsof a light chain variable region of a murine anti-CD74 mAb, thatcomprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQID NO:13), CDR2 comprising an amino acid sequence TVSNRFS (SEQ IDNO:14), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ IDNO:15), and the heavy chain variable region of the humanized mAbcomprises CDRs of a heavy chain variable region of a murine anti-CD74mAb, that comprises CDR1 comprising an amino acid sequence NYGVN (SEQ IDNO:16), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ IDNO:17), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ IDNO:18).

The CDRs of the light chain variable region of the humanized PAM4 MAbcomprise CDR1 comprising an amino acid sequence of SASSSVSSSYLY (SEQ IDNO:19); CDR2 comprising an amino acid sequence of STSNLAS (SEQ IDNO:20); and CDR3 comprising an amino acid sequence of HQWNRYPYT (SEQ IDNO:21); and the CDRs of the heavy chain variable region of the humanizedPAM4 MAb comprise CDR1 comprising an amino acid sequence of SYVLH (SEQID NO:22); CDR2 comprising an amino acid sequence of YINPYNDGTQYNEKFKG(SEQ ID NO:23) and CDR3 comprising an amino acid sequence of GFGGSYGFAY(SEQ ID NO:24).

The CDRs of the light chain variable region of the humanized anti-CSApMAb comprises CDR1 comprising an amino acid sequence of RSSQSIVHSNGNTYLE(SEQ ID NO:25); CDR2 comprising an amino acid sequence of KVSNRF (SEQ IDNO:26) and CDR3 comprising an amino acid sequence of FQGSRVPYT (SEQ IDNO:27); and the CDRs of the heavy chain variable region of the humanizedanti-CSAp MAb comprises CDR1 comprising an amino acid sequence of EYVIT(SEQ ID NO:28); CDR2 comprising an amino acid sequence ofEIYPGSGSTSYNEKFK (SEQ ID NO:29) and CDR3 comprising an amino acidsequence of EDL (SEQ ID NO:30).

What is claimed is:
 1. An immunoconjugate comprising: a) an anti-EGP-1antibody or an antigen-binding fragment thereof; and b) six or moremolecules of SN-38 covalently attached to the antibody or antibodyfragment, wherein the attachment between SN-38 and antibody or antibodyfragment involves a covalent bond with the C-20 hydroxyl group of theSN-38.
 2. The immunoconjugate of claim 1, wherein the antibody is achimeric, humanized or human antibody.
 3. The immunoconjugate of claim1, further comprising a linker between the antibody or antibody fragmentand the SN-38.
 4. The immunoconjugate of claim 3, wherein the linker isan intracellularly cleavable linker.
 5. The immunoconjugate of claim 1,wherein the antibody is hRS7.
 6. The immunoconjugate of claim 1, whereinthe antibody fragment is selected from the group consisting of a Fab,Fab′, F(ab)₂, F(ab′)₂ and scFv antibody fragment.
 7. The immunoconjugateaccording to claim 1, wherein said antibody or antibody fragmentcomprises light chain variable region complementarity determining region(CDR) sequences CDR1 (KASQDVSIAVA, SEQ ID NO:7), CDR2 (SASYRYT, SEQ IDNO:8) and CDR3 (QQHYITPLT, SEQ ID NO:9) and heavy chain variable regionCDR sequences CDR1 (NYGMN, SEQ ID NO:10), CDR2 (WINTYTGEPTYTDDFKG, SEQID NO:11) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:12).
 8. An immunoconjugatecomprising: a) an anti-EGP-1 antibody or antigen-binding fragmentthereof; and b) at least one molecule of SN-38 covalently attached tothe antibody or antibody fragment, wherein the attachment between SN-38and antibody or antibody fragment involves a covalent bond with the C-20hydroxyl group of the SN-38.
 9. The immunoconjugate of claim 8 whereinthe antibody is RS7.
 10. The immunoconjugate of claim 8, wherein theantibody or antibody fragment comprises light chain variable region CDRsequences CDR1 (KASQDVSIAVA, SEQ ID NO:7), CDR2 (SASYRYT, SEQ ID NO:8)and CDR3 (QQHYITPLT, SEQ ID NO:9) and heavy chain variable region CDRsequences CDR1 (NYGMN, SEQ ID NO:10), CDR2 (WINTYTGEPTYTDDFKG, SEQ IDNO:11) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:12).
 11. The immunoconjugate ofclaim 8, wherein the antibody is a humanized antibody.